Induction of IL-12 using immunotherapy

ABSTRACT

The present invention relates to compositions and methods that promote the induction of IL-12 in a patient. The composition includes activated allogeneic cells that are administered to a patient with a disease such as cancer. Administration of the composition skews the patient&#39;s immune response to a Th1 environment and produces detectable levels of IL-12 in the patient&#39;s plasma, without any IL-12 related toxicity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/581,745, filed Nov. 9, 2012, which is a Section 371 National StageApplication of International Application No. PCT/US2012/036123, filedMay 2, 2012, in English, which claims priority to U.S. provisionalpatent application Ser. No. 61/482,009, filed May 3, 2011, U.S.provisional patent application Ser. No. 61/528,484, filed Aug. 29, 2011,U.S. provisional patent application Ser. No. 61/564,551, filed Nov. 29,2011 and U.S. provisional patent application Ser. No. 61/582,881, filedJan. 4, 2012, the contents of each are hereby incorporated by referencein their entirety.

FIELD

This invention relates to therapy using immune cells. More specifically,this invention relates to immune cell therapy that promotes IL-12production in patients.

BACKGROUND

The most precise, powerful and safest disease prevention and treatmentmechanism known is the natural ‘sterilizing’ immune response whichcombines elements of both innate and adaptive immunity to clear the bodyof a large variety of foreign pathogens without medical intervention.The immune system is designed to ‘remember’ the cleared foreign antigensin order to quickly mount an immune response upon re-infection. Immunesystems, even those of cancer patients, can recognize and mount aresponse to foreign antigens, such as found on viruses and bacteria,sufficiently enough to completely destroy and eliminate them from thebody. The ferocity and specificity of this sterilizing immune responsecan be witnessed in the manner in which an inadequately suppressedimmune system can completely destroy large transplanted organs, such asa kidney, liver or heart, while sparing self tissues. The destructiveeffect of this immunity against foreign antigens would be beneficial ifthis effect could be redirected to tumors and/or other antigens thatescape due to an insufficient immune response by the patient.

Immunotherapy is dedicated to developing methods to harness, direct andcontrol the immune response against a variety of infectious andnoninfectious diseases including cancer. Therapeutic vaccines are a typeof immunotherapy designed to educate the immune system. In patients withexisting cancers, the vaccines are designed so the patient's immunesystem recognizes the tumor cells as foreign. If tumors are recognizedby the immune system as a foreign pathogen, an immune response couldtheoretically be elicited which could cause immune cells to destroylarge tumors and seek out and destroy metastatic tumor cells whereverthey reside in the body. After successful immunotherapy, the ability ofthe immune system to ‘remember’ eliminated foreign cells would enablethe immune system to eliminate any recurrent cancer cells without anyadditional treatment, much like the immune system protects againstopportunistic infections.

An individual's immune system response to diseases or to diseaseorganisms can be either a Th1 response or Th2 response. In a Th1response, the CD4+ T cells become polarized toward Th1 cells andconversely, in a Th2 response, the CD4+ T cells become polarized towardTh2 cells. This increasingly popular classification method is referredto as the Th1/Th2 balance. Th1 cells promote cell-mediated immunity,while Th2 cells induce humoral immunity. Cellular immunity (Th1) directsnatural killer cells (NK), T-cells and macrophages to attack abnormalcells and microorganisms at sites of infection. Humoral immunity (Th2)results in the production of antibodies used to neutralize foreigninvaders. In general, Th2 polarization of CD4+ T cells has been shown torelate to cancer progression in most human and animal cancer studies,while Th1 polarization is correlated with tumor regression andanti-tumor immunity.

The immune response of an individual, Th1/Th2 balance, can be evaluatedthrough the balance of cytokines in the individual. Cytokines are smallcell-signaling protein molecules. The term cytokine is used as a genericname for a diverse group of soluble proteins and peptides that act asregulators normally at nano- to picomolar concentrations and which,either under normal or pathological conditions, modulate the functionalactivities of individual cells and tissues. These proteins also mediateinteractions between cells directly and regulate processes taking placein the extracellular environment. Interleukins are a group of cytokinesinvolved in immunomodulation and can be synthesized by a variety ofcells in the immune system. There are a number of interleukins, such asIL-2, IL-4, IL-10 and IL-12, and each of these interleukins has aspecific role within the immune system.

Th1 cells produce Type 1 cytokines that are involved in inflammatoryresponses. Type 1 cytokines include, for example, IL-2, IL-12, IL-15,WN-gamma, TNF-alpha, TNF-beta, GM-CSF and C-C chemokines. Th2 cellsproduce Type 2 cytokines that are involved in humoral immune responses.Type 2 cytokines include, for example, IL-4, IL-5, IL-6, IL-10, IL-13and TGF-beta. Th1 and Th2 immune responses are counter-regulatory, suchthat increased Type 1 responses downregulate Type 2 responses andincreased Type 2 responses downregulate Type 1 responses.

IL-12 is a heterodimer composed of a p35 and a p40 subunit. It isproduced primarily by Antigen Presenting Cells (APC). IL-12 can also beproduced by monocytes and macrophages, dendritic cells and B-cells.IL-12 exerts immunomodulatory effects on T-cells and natural killercells. Endogenous IL-12 is known to be involved in generating optimalTh1 responses and can play an important role in cell-mediated immunityagainst intracellular pathogens.

IL-12 has been the subject of intense investigation because it modulatesimportant components of the immune system and has been demonstrated tohave dramatic anti-tumor effects in the laboratory and in animalstudies. IL-12 has been implicated, for example, in inhibiting growth ofhuman lung adenocarcinoma and acute myeloid leukemia. However, the useof exogenous IL-12 in a therapeutic regimen has been limited by hightoxicity in humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the IL-12 level in a patient's plasmaover more than a year. Allogeneic, activated Th-1 cells wereadministered to the patient at various times using various modes ofadministration.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to compositions and methods that lead todetectable levels of IL-12 in the plasma of a patient. The presentinvention includes a composition that, when administered to a patient,can lead to the production of detectable levels of endogenous IL-12 inthe patient's plasma, without any significant toxicity. The endogenousIL-12 can surprisingly be detected in patients with cancer. Thecomposition preferably includes allogeneic activated T-cells. T-cellsare not capable of producing IL-12, therefore the T-cell compositionadministered to the patient elicits the production of IL-12 by thepatient's own APC.

The present invention also includes methods of inducing production ofendogenous IL-12 in a patient by the patient's own immune system. Themethod includes administering a composition of allogeneic material,preferably allogeneic activated T-cells. The composition may beadministered as a single dose or multiple doses. Preferably, theallogeneic activated T-cells are administered in frequent, low doses.The allogeneic cells can be administered by intradermal, intravenous orintralesional routes. Preferably, the frequency is not less than every 3days. When these compositions are administered, the patient's own immunesystem can be induced to produce detectable levels of endogenous IL-12in the plasma, even in a patient with a cancer. Generally, IL-12 is notfound in patients with cancer because tumors can inhibit expression ofIL-12. Surprisingly, the methods described herein can overcome thisinhibition and create an environment sufficient to induce expression ofIL-12 in the plasma for extended periods of time, for example, severalmonths or even a year. Furthermore, the presence of endogenous IL-12 inplasma does not lead to significant toxicity in the patient as does theadministration of exogenous IL-12 as medicant.

By endogenous IL-12, it is meant that the IL-12 is synthesized in thepatient by the patient's own immune system. Specifically, the IL-12 canbe synthesized by the patient's antigen presenting cells (APC). APC caninclude monocytes and macrophages, dendritic cells and B-cells. Byexogenous IL-12, it is meant that the IL-12 is not synthesized by thepatient's own immune system. Exogenous IL-12 includes IL-12 isolatedand/or purified IL-12 from another individual or IL-12 that is expressedby a DNA construct(s) that includes a gene for IL-12.

Advantageously, the systemic production of endogenous IL-12 in thepatient leads to minimal or no toxicity to the patient. The patient mayexperience transient symptoms such as transient flu-like symptoms.Generally, when exogenous IL-12 has been administered to the patient,toxic effects have limited the use in a therapeutic setting. The abilityof the methods described herein to promote endogenous production ofIL-12 that can lead to systemically detectable levels of IL-12 in theplasma without toxicity is surprising. This result enables the use ofthe patient's own immune system to harness the benefits created from thepresence of IL-12 toward reduction and/or elimination of tumors andcancerous cells.

The use of these methods can also be applicable to reduction and/orelimination of other diseases that respond favorably to a Th1environment, specifically to IL-12. Such diseases include cancer,infectious diseases, including chronic viral and intracellular bacterialor mycobacterial diseases, such as hepatitis B, hepatitis C, HIV1, HIV2,HTLV1, HTLV2, HPV, mycobacterium tuberculosis, periodontal disease, andallergic diseases like atopic asthma. In addition, methods to promotethe endogenous production of IL-12 can have an anti-aging effects bymaintaining cellular immunity. The balance of Th1 to Th2 cells in normalindividuals decreases as part of the aging process, making the elderlymore susceptible to infectious diseases and cancer. Promotion ofendogenous IL-12 production can increase the Th1/Th2 ratio, thusprotecting against vulnerability to disease.

The compositions of the present invention generally include foreignantigens, preferably alloantigens. The compositions also include atleast one Th1 cytokine and/or at least one DC effector molecule capableof inducing the maturation of DC to produce IL-12. The therapeuticcomposition generally includes the at least one Th1 cytokine, and/or theat least one DC effector molecule combined together with thealloantigen. The composition preferably contains living allogeneicactivated T-cells that are capable of providing each of the componentsof the composition in a single cell type. In preferred embodiments,living allogeneic Th1 cells that are activated to produce Th1 cytokines,such as interferon-gamma, tumor necrosis factor-alpha and interleukin-2and express the DC maturation effector molecule CD40L on the cellsurface are used. Alternatively, the three components of the compositioncould be sourced from more than one cell type. For example, the Th1cytokines may be sourced from one cell type in a composition and thealloantigen from a separate cell type and the DC effector molecules froma third cell type. Alternatively one cell type could contain any two ofthe components and a second cell type contain the third. The cell typesdo not need to be living as long as they provide a source of thenecessary components of the composition.

Alternatively, the composition components can be sourced from natural orbioengineered proteins. For example, recombinant or purified Th1cytokines or DC maturation molecules or alloantigens could be usedtogether or in combination with living cell components. The compositioncomponents could be combined on a “chip” or biodegradable platform. Thecomponents do not need to be delivered at the same time to a patient,but can be delivered in any sequence.

The alloantigens in the therapeutic compositions must be provided in amanner that the antigen can be engulfed or presented to the immunesystem in order to be processed and presented to T-cells. The antigencan be a natural part of living cells or can be altered or bioengineeredusing molecular biological techniques. The antigen can be soluble orimmobilized on a surface, an intact part of a living organism or cell,or a part of an attenuated organism. In preferred embodiments, thealloantigens are allogeneic T-cells and in more preferred embodiments,allogeneic activated T-cells.

In one exemplary embodiment, the therapeutic composition includesalloantigens expressed on T-cells. The T-cells are preferably CD4+T-cells, and more preferably Th1 cells. The Th1 cells can be in-vitrodifferentiated, expanded and activated from naïve CD4+ precursor cellsderived from normal blood donors. Preferably, the cells are in anactivated state at the time of administration. Preferably, the cells areactivated by cross-linking monoclonal antibodies directed to CD3/CD28surface molecules. Crosslinking is preferably caused by immobilizationof the CD3/CD28 monoclonal antibodies on a surface. Preferably, thesurface is a micro- or nanabead particle. The beads may be biodegradablebeads. These cells can produce large amounts of inflammatory Th1cytokines and express effector molecules on the cell surface, such asCD40L, which serve to promote the development of Th1 immunity by causingendogenous IL-12 production.

In preferred embodiments, the therapeutic composition includes activatedallogeneic Th1 cells. These activated Th1 cells can be powerfulinflammatory agents. These activated allogeneic Th1 cells and methodsfor preparing them are described, for example, in U.S. Pat. Nos.7,435,592, 7,678,572, 7,402,431 and 7,592,431 and are incorporatedherein by reference. The activated allogeneic Th1 cells areintentionally mismatched to the patient.

A variety of Th1 inflammatory cytokines may be included in thetherapeutic compositions. Examples of inflammatory Th1 cytokinesinclude: IL-1, IL-2, IL-6, IL-12, IL-15, IFN-gamma, TNF-alpha, TNF-beta,GM-CSF and C-C chemokines and do not include TGF-beta, IL-4 or IL-10.The cytokine component can be natural or recombinant cytokines or can bebioengineered molecules designed to interact with the receptors for acytokine. The cytokines may be directly included in the therapeuticcompositions. Alternatively, the therapeutic compositions can includeliving cells or other components that produce and secrete the cytokines.Preferably the cytokines are provided naturally through an activatedcell source, as exogenous cytokines tend to be very toxic to patientswhile endogenous cytokines are not. In some exemplary embodiments, thetherapeutic compositions include T-cells in an activated state that areproducing and secreting the inflammatory Th1 cytokines and thus, canserve as the source of these cytokines in the therapeutic compositions.

The therapeutic composition can include a factor or factors that causethe maturation of immature DCs. Specifically, maturation factors whichpromote DC1 cell maturation and IL-12 production leading tointerferon-gamma production and Th1 adaptive immunity. DCs are capableof evolving from immature, antigen-capturing cells to mature,antigen-presenting, T cell-priming cells which convert antigens intoimmunogens and express cytokines, chemokines, costimulatory moleculesnecessary to initiate an immune response. The types of T cell-mediatedimmune responses (Th1 vs. Th2) induced varies depending on theactivation signals received from the surrounding microenvironment. Theability of DCs to regulate immunity such as anti-tumor andanti-infectious disease immunity is dependent on DC maturation topromote Th1 immunity. Human DCs are not a homogenous population. Besidesinducing anti-tumor immunity, DCs can induce anergy or tolerance. DCsoriginate from CD34+ hematopoietic stem cells (HSC). Myeloid dendriticcells (DC1) and plasmacytoid DCs (DC2) are the two principalsubpopulations of human DCs, and their characteristics vary greatly inphenotype, migration, and function. DC1 cells are effective T cellstimulators, inducing a tumor specific immune response. CD11c+DC1 cellsprimarily induce Th1 differentiation, whereas DC2 cells, which expressthe receptor for IL-3 (CD123), mainly promote a Th2 response. Both DCpopulations are significantly lower in patients with cancer than inhealthy donors. DC1 cells produce IL-12 upon maturation and DC2 cellsproduce IL-10.

Production of cytokines such as IL-10 and IL-12 during the DC maturationprocess influences DC induction of a Th1 or Th2 immune response. Inaddition to expressing high levels of antigen-presenting molecules andcostimulatory molecules, mature DC must release large amounts of IL-12in order to stimulate a Th1 immune response. Release of IL-10, blocksthe DC maturation process by interfering with up-regulation ofcostimulatory molecules and production of IL-12, subsequently limitingthe ability of DCs to initiate a Th1 response.

A variety of factors can induce maturation of DC to become DC1, IL-12producing cells following antigen uptake and processing, including:whole bacteria or bacterial-derived antigens (e.g. lipopolysaccharide,LPS), inflammatory cytokines such as IFN-gamma, TNF-alpha, IL-1, GM-CSF,ligation of select cell surface receptors (e.g. CD40), viral products(e.g. double-stranded RNA), Fas engagement on immature DCs, for example,induces both maturation and release of IL-1 beta and IFN-gamma. Ligationof CD40 promotes an up-regulation of the costimulatory moleculesB7-1/CD80 and B7-2/CD86 and IL-12 secretion and release of chemokines(e.g. IL-8, MIP-1 alpha, MIP-1 beta).

In some preferred embodiments, CD40L is included as a factor formaturation of the DCs. Inclusion of other factors that cause maturationof the DCs is also within the scope of the invention. In some exemplaryembodiments, the therapeutic compositions include T-cells in anactivated state which express high density CD40L on the surface. CD40Lis a potent effector molecule for DC maturation to produce IL-12.

In one exemplary embodiment, the therapeutic composition includesactivated allogeneic T-cells, at least one type I cytokine and at leastone factor that causes maturation of DCs. Compositions including thesecomponents are described, for example, in pending U.S. patentapplication Ser. No. 12/967,910 filed on Dec. 14, 2010 and incorporatedherein by reference.

Intratumoral administration of the therapeutic compositions afterablation of some of the tumor cells in order to release tumor associatedantigens into the microenvironment can provide a potent adjuvant effectfor the maturation of DC to DC1 phenotype which produces IL-12 andpromotes development of Type 1 anti-tumor immunity and the downregulation of tumor immunoavoidance mechanisms. Administration of thetherapeutic composition can also be accomplished by other methodsincluding, for example, intravenous, intradermal, intrathecal,intraperitoneal, intralesional, intrapleural administration and thelike. Preferably, the composition is first administered intradermally,as the skin is rich in immature DC called Langerhans cells. In thepresence of inflammatory Th1 cytokines, such as interferon-gamma, tumornecrosis factor-alpha, IL-2 and GM-CSF and a DC maturation factor, suchas CD40L, the Langerhan's cells uptake the alloantigen and mature toDC1, IL-12 producing cells. These mature cells migrate to the lymphnodesand promote development of Th1 immunity.

Intradermal injections of the composition can “prime” a patient tobecome immune to the alloantigen in the composition. Multipleintradermal injections can increase the number of Th1 memory cellsspecific for the alloantigens in the circulation of the patient, whichin turn changes the Th1/Th2 balance. Injection of 1×10⁶ cells to 1×10⁷allogeneic activated Th1 cells is a preferred intradermal dose, 1×10⁷cells in 1 ml of fluid is the most preferred. The intradermal dosing ispreferably repeated multiple times in order to build up the number ofcirculating Th1 memory cells. The intradermal dosing frequency ispreferably about 3-4 injections every 7 days, more preferably every 3-4days.

In preferred embodiments, the intradermal dosing is followed by anintratumoral dosing of the composition to create an in-situ vaccine. Theintratumoral dosing is preferably conducted following the in-situablation of some of the tumor cells in the target lesion. The ablationis preferably caused by use of extreme cold (cryoablation) or heat(radiation), but can be also done using a variety of methods includingalcohol ablation, chemotherapy and/or monoclonal antibody drugs. Apreferred intratumoral dose is between about 1×10⁷ and 1×10⁸ cells, mostpreferably about 3×10⁷ cells. It is preferred that a first intratumoraldose be injected immediately following the ablation and a second withinabout 7 days, preferably within about 3-4 days following the firstinjection. This process of ablation followed by intratumoral injectionof the composition can be repeated as necessary.

The method also preferably includes administering the compositionintravenously in order to cause the activation of host immune cells(both innate and adaptive) and their extravasation to sites ofinflammation, including tumor locations. The intravenous dose of thecomposition of allogeneic activated Th1 cells preferably includes about1×10⁷ to 1×10⁹ cells, more preferably about 5×10⁷ to 1×10⁸ cells. Theintravenous infusions can be repeated several times, preferably on amonthly basis.

The allogeneic Th1 cells of the composition preferably produce largeamounts of the Type 1 cytokines: IL2, IFN-γ, TNF-alpha □ and GM-CSF. Thepresence of inflammatory Th1 cytokines in a microenvironment whereimmature DC are engulfing and processing antigens can help promotematuration to DC1, IL-12 producing DC. IL-12 can stimulate the level ofIFN-γ that in turn can lead to promotion of a Th1 immunity. IFN-γ is apivotal Type 1 cytokine necessary to promote Type 1 anti-tumor immunity.IFN-γ can mediate anti-tumor effects by directly inhibiting tumor cellgrowth and inducing T cell-mediated anti-tumor responses. IFN-γsecretion can independently contribute to the NK cell response andenhance the NK cell response activated by IL-12.

The preferred medicament containing activated allogeneic Th1 cells canbe derived from precursors purified from normal, screened blood donors.The cells should be supplied as a sterile, low endotoxin dosage formformulated for either intradermal or intratumoral injection, orintravenous infusion. The cells may also be formulated forintraperitoneal, intrapleural or epidural infusions. The donors arepreferably tested to be negative for HIV1, HIV2, HTLV1, HTLV2, HBV, HCV,RPR (syphilis), and the cells are preferably tested to be negative formycoplasma, EBV and CMV. In preferred embodiments, the activatedallogeneic cells are HLA mismatched with the patient.

The methods of the present invention generally relate to producingdetectable levels of endogenous IL-12 in the patient's plasma. Themethods include administering the compositions of the present inventionin such a way as to engineer the patient's immune system to produceendogenous IL-12 at detectable levels in the patient's plasma. Themethods described herein can increase the circulating numbers of Th1immune cells in cancer patients, shifting the balance from Th2environment to a Th1 environment. Additionally, the methods may alsoinclude steps that elicit an anti-tumor specific Th1 immunity and/oractivate components of the innate and adaptive immune responses togenerate a sustained Th1 cytokine environment in order to down-regulatetumor immunoavoidance.

The methods of the present invention can include administering acomposition containing a foreign antigen to promote Th1 immunity in thepatient against the foreign antigen. The method may also includeablating all or a portion of the tumor that results in at least sometumor necrosis. A variety of methods can be used to generate tumornecrosis in the patient. The method may also involve creating aninflammatory microenvironment in proximity to the site of tumornecrosis, i.e the site of the tumor lesion. In addition, the method canalso include activating the adaptive and innate immune cells of thepatient to maintain a prolonged. Th1 environment. In preferredembodiments, a key component of the method includes the use of amedicant or composition containing activated allogeneic T cells asdescribed above.

Since most human cancer patients generally present with polarized Th2immunity, the objective of this method of treatment is generally toincrease the amount of circulating Th1 cells in cancer patients. Thenumber of circulating Th1 cells can be built up in the cancer patient byadministering one of the therapeutic compositions described above to thepatient that includes a foreign antigen.

In an exemplary embodiment, the patient is administered activatedallogeneic Th1 cells that are injected intradermally. In preferredembodiments, intradermal injections are on a weekly schedule once a weekfor about 3-4 weeks. In other preferred embodiments, the intradermalinjections may be administered multiple times about every 3-4 days.Intradermal injections may be administered every two days or up to ayear apart. The injection schedule should be designed to enhance thefootprint of Th1 memory cells in circulation. The alloantigens expressedon the foreign cells can stimulate a potent immune rejection response.In addition, the presence of Th1 cytokines in the composition or theexpression of Th1 cytokines by the allogeneic cells can provide theinflammatory adjuvant environment necessary to steer the immune responseto the alloantigens toward Th1 memory immunity. This can create anincreased pool of Th1 memory cells in circulation in the patientspecific for the alloantigens contained within the allogeneic Th1 cells.Multiple administrations can act as booster shots, increasing the numberof circulating memory Th1 cells specific for the alloantigens.

In some embodiments, the administration of allogeneic activated T cellsmay be followed by additional steps to enhance the patient response.These steps can include, for example, ablation of the tumor that causestumor necrosis along with intratumoral administration of additionalallogeneic activated T cells. Additional administration of theallogeneic activated cells intravenously may also be performed. Thesemethods are described in the U.S. Pat. No. 7,972,594 to Har-Noyincorporated herein by reference.

The administration of the therapeutic compositions or medicaments usingthe methods described herein can promote the systemic production ofendogenous IL-12 in the patient by the patient's own immune system. Theconcentration of the endogenous IL-12 in the patient is sufficient thatthe IL-12 can be detected in the patient's plasma. The detectable levelsof IL-12 are endogenous and not a result of any that might be present inthe therapeutic composition because generally the components of thecomposition are eliminated by the patient's immune system in therejection response elicited by the administration of allogeneicmaterial. In preferred embodiments, the composition contains T-cellswhich can not produce IL-12. Thus, any of the IL-12 detected in thepatient's plasma is a result of the IL-12 produced by the patient's ownimmune system.

Preferably, the IL-12 is produced by the patient's immune cells, forexample, the patient's own monocytes, natural killer cells and dendriticcells. These cells will have matured under the influence of theinflammatory or Type I cytokines generated by the administration of thecompositions described herein.

The concentration of the IL-12 in the patient's plasma can vary but isgenerally at least about 8000 pg/ml. The concentration of the IL-12 inthe patient's plasma is preferably between about 8000 pg/ml to 200,000pg/ml. As described herein, the concentration of the IL-12 detected inthe plasma of the patient does not lead to toxicity issues. However,administration of exogenous IL-12 has been known to be toxic topatients. Patients that seroconvert to IL-12 expression in the plasmahave an increased survival compared to patients that do not expressIL-12 in their serum. The level of IL-12 may not correlate withsurvival, only the presence of IL-12 is crucial.

The increase in IL-12 is generally detected after a period of time afterthe administration of the composition. Preferably, after about 3-4 weeksof dosing with therapeutic composition, the IL-12 can be detected in theplasma. There can be a delay in IL-12 seroconversion for about 90-120days after the administration of the last composition.

The IL-12 in the plasma can be detected by using a variety of methods.IL-12 has two subunits called the p40 and p35 chains and antibodiesspecific to p40 are preferred for detection. Several methods areavailable to detect the presence of IL-12. Detection of IL-12 caninclude, for example, ELISA, and cytokine bead array.

The methods described herein can be suitable for a variety of patients,including humans. The methods may also be used on other mammals.

The present invention also includes methods of treating a disease in apatient. The diseases can include cancerous tumors as described above,hematological malignancies, as well as diseases caused by pathogenicagents. Other diseases that are susceptible to a Th1 response in apatient can also be treated using the methods described herein. Thepatient is administered the allogeneic composition according to themethods described herein. The patient's plasma is then monitored for thepresence of IL-12. The detection of endogenous IL-12 can be indicativeof the patient's immune response to the disease. Additionaladministrations of the therapeutic composition may be performed formaintenance of the IL-12 levels and thereby maintaining the patient'simmune response against the disease antigens.

Examples

This study was performed to monitor the level of IL-12 in the plasma ofa patient treated with allogeneic, activated Th1 cells. These activatedallogeneic Th1 cells and methods for preparing them are described inU.S. Pat. No. 7,435,592. The activated allogeneic Th1 cells wereintentionally mismatched to the patient.

Intradermal Injections-Intradermal injections of activated allogeneicTh1 cells were administered. The cells were suspended in 1 ml at adensity of 1×10⁷ cells/ml.

Intratumoral Injections-Intratumoral injection was administered in thenecrotic center of an ablated tumor within one hour of ablation.

Cryoablation was done with the use of a CryoCare-28 Percutaneous ProbeSystem (Endocare, CA, USA). This system used the Joule-Thomson effect tocool the end of a cryoprobe in a closed system. In accordance with thegas coefficient and the dimension of the nozzle, different gaseouselements generate different thermal exchange events at the area close tothe nozzle. Argon gas was used for cooling (−187° C.), and helium wasused for heating (67° C.).

The planned target tumor lesion was identified and located under CTimage guidance. A sterile field was created and local anesthesiaadministered to the planned probe insertion site. A guide probe wasinserted percutaneously and verified by CT to be within the target tumorlesion. One or two freeze-thaw cycles were performed. A single probe of2- or 5-mm was used according to the size of the target tumor. The timeof freezing was approximately 5-20 minutes dependent on the achievementof an “ice-ball”, visible on CT. Thawing was achieved by input of heliumduring a period equivalent to the freezing time before the secondfreezing process was initiated. The procedure requires ablation of asample of the tumor lesion and does not require complete tumor ablationwith tumor-free margins.

The lesion was allowed to cool following the second freezing cyclebefore injection of the allogeneic activated Th1 cells.

Tables 1, 2 and 3 show the timing of the specific treatments and thelevel of IL-12 in the patient's plasma on the indicated days. FIG. 1 isa graph illustrating the IL-12 expression by the patient's immune systemduring the study.

TABLE 1 pt#11 weeks from BL day from BL treatment IL12 pg/ml 0 0 Base 0 3 w + 2 d 23 Cryo + IT + IV 0  4 w + 2 d 30 post-2nd IV 0 14 w + 2 d100 post-IV-B 0 16 w + 1 d 113 pre-1st ID in ID/IV prot. 11,317 136post-3rd IV in ID/IV prot. 50,725 21 w + 1 d 148 pre-IV-B in ID/IV prot.64,117 149 post-IV-B in ID/IV prot. 60,301 25 w + 2 d 177 Pre-IV-B 60 DIV/ID (T) 151,048 14 APR. 2010 178 Post-IV-B 60 D (T) 88,362 15 APR.2010 179 48 h Post-IV-B 60 D (T) 135,169 16 APR. 2010 180 72 h Post-IV-B60 D (T) 79,476 17 APR. 2010 27 w + 3 d 192 F/U 14,840 197 Pre-IV-B8,867 198 Post-IV-B 10,610 37 w + 3 d 262 F/U D240 32,188 42 w + 3 d 297pre-chemo 35,115 46 w + 3 d 325 35,552 49 w + 3 d 346 16,265 50 w + 3 d353 15,584 52 w + 1 d 365 16,546 52 w + 6 d 370 Plasma* 22,626

TABLE 2 treatment day from BL IL12 pg/ml Base 0 0 Cryo + IT + IV 23 0post-2nd IV 30 0 post-IV-B 100 0 pre-1st ID in ID/TV prot. 113 11,317post-3rd IV in ID/IV prot. 136 50,725 pre-IV-B in ID/IV prot. 148 64,117post-IV-B in ID/IV prot. 149 60,301 Pre-IV-B 60 D IV/ID (T) 177 151,04814 APR. 2010 Post-IV-B 60 D (T) 178 88,362 15 APR. 2010 48 h Post-IV-B60 D (T) 179 135,169 16 APR. 2010 72 h Post-IV-B 60 D (T) 180 79,476 17APR. 2010 F/U 192 14,840 Pre-IV-B 197 8,867 Post-IV-B 198 10,610 F/UD240 262 32,188 pre-chemo 297 35,115 325 35,552 346 16,265 353 15,584365 16,546 pre-IV-B 370 22,626 F/U 374 26,405 F/U 388 219,275 F/U 390155,023 F/U 394 336,141 F/U 401 113,513 F/U 408 92,122 F/U 417 63,357F/U 423 79,075 F/U 429 48,038 F/U 436 59,471

TABLE 3 Chemo days fromBL 1st 296 start 301 stop 2nd 324 start 331 stop3^(rd) 345 start 352 stop 4^(th) 359 start 364 stop 5^(th) 408 start 415422 stop 6^(th) 436 start

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. A composition capable of causing the appearanceof IL-12 in plasma, the composition essentially consisting of: a foreignantigen; at least one Th1 cytokine; and a DC maturation molecule.
 2. Thecomposition of claim 1 where the foreign antigen is an alloantigen. 3.The composition of claim 1 where the Th1 cytokine is one or more of thefollowing: IL-1, IL-2, IL-6, IL-8, IL-15, Interferon-gamma, TNF-alpha,GM-CSF.
 4. The composition of claim 1 where the DC maturation moleculeis CD40L and/or FasL.
 5. The composition of claim 2 where thealloantigen is on a living T-cell.
 6. The composition of claim 5 wherethe T-cells are CD4+ cells or Th1 cells.
 7. The composition of claim 6where the Th1 cell is activated.
 8. The composition of claim 7 where theTh1 cells are activated by cross-linking CD3 and CD28.
 9. Thecomposition of claim 8 where the activated Th1 cells secrete one or moreof the following Th1 cytokines: IL-2, IFN-gamma and GM-CSF.
 10. Thecomposition of claim 9 where the activated Th1 cells express the DCmaturation molecule CD40L and/or FasL on their surface.
 11. Thecomposition of claim 1 where the components are immobilized on asurface.
 12. The composition of claim 11 where the surface isbiodegradable.
 13. The composition of claim 5 where the living T-cellsare packaged in a syringe or flexible container.
 14. The composition ofclaim 13 where the cells are at a concentration of 1×10⁷ cells/ml orgreater.
 15. The composition of claim 14 where the cells are suspendedin a non-nutrient media.
 16. A method of treating a patient with adisease comprising: administering a composition comprising allogeneiccells wherein at least a portion are activated T-cells, wherein theT-cells are activated by cross-linking one or more agents bound to thecell surface moieties on the T-cells; monitoring the level of IL-12 inthe patient's plasma; and readministering the composition if IL-12 isnot detected in the plasma of the patient.
 17. The method of claim 16wherein the disease is cancer or is a result of a pathogenic infection.18. The method of claim 16 wherein the readministering is repeated untilthe concentration of the endogenous IL-12 is detectable.
 19. Atherapeutic composition for increasing the endogenous levels of IL-12comprising activated allogeneic T cells wherein administration of thecomposition leads to detectable levels of endogenous IL-12 in the plasmaof a patient without toxicity to the patient.
 20. The composition ofclaim 19 wherein the patient has cancer or an infectious disease. 21.The composition of claim 19 wherein the T-cells are CD4+ cells.
 22. Thecomposition of claim 19 wherein the T-cells are activated bycross-linking one or more agents that are bound to the cell surfacemoieties on the T-cells.
 23. The composition of claim 19 wherein the oneor more agents are monoclonal antibodies such as anti-CD3 and anti-CD28monoclonal antibodies.